Stimulation of T cell receptors causes depletion of intracellular Ca2+ stores and subsequent opening of the CRAC (Ca2+-release-activated Ca2+) channels. A sustained increase in intracellular Ca2+ concentration activates the calcineurin/NFAT (nuclear factor of activated T cells) pathway and turns on transcriptional programs of various cytokines. Recently, Orai1 and STIM1 were identified as a long-sought pore component of CRAC channels and as an endoplasmic reticulum (ER) Ca2+ sensor, respectively. STIM1 senses Ca2+ depletion in ER after stimulation of T cell receptors, translocates to plasma membrane (PM) proximal ER, binds to and activates Orai1. Human patients deficient in Orai1 or STIM1 have severe combined immune deficiency. However, this deficiency has been rescued by hematopoietic stem cell transplantation, implying an irrevocable role of Orai1 specifically in the immune cells. Thus, a specific blocker of Orai1 is likely to ameliorate the side effects observed with calcineurin blockers. Here, we propose to develop chemical library screens for identification of small molecule blockers for CRAC channels. To identify specific blockers for Orai1 using high throughput screening, we have tested more than 85,000 chemical compounds in a primary screen. Strong candidates were validated in secondary screens. The short-term specific aims are: (1) To validate the specificity and efficacy of Orai1 blockers. To determine specificity and efficacy of the blockers, the efficacious dose of the candidate compounds will be determined in T cells. Tertiary screening efforts will utilize whole-cell patch clamping and total internal reflection fluorescence microscopy to understand the molecular mechanism of inhibition by the candidate drugs. (2) To test the efficacy of immunosuppression by the candidate blockers in clinically relevant animal models. To test the candidate molecule's therapeutic potency, we plan to determine whether the injection of the candidate compounds ameliorate autoimmune diseases using animal models. Development of small molecule blockers of CRAC channels would represent a novel and improved therapeutic approach to suppress immune response during organ transplant and autoimmune diseases. Such small molecule blockers targeting Orai1 activity is likely to have much less side effects than immunosuppressive drugs such as cyclosporin A and tacrolimus because the CRAC channel is specifically predominant in immune cells.